Culture optimization of Streptomyces sp. KRA16-334 for increased yield of new herbicide 334-W4

This study aimed to isolate actinomycetes that exhibit strong herbicidal activity, identify compounds active against weeds, and researching methods to improve the production of these compounds through culture optimization to establish a foundation for the development of environmentally friendly bioherbicides. 334-W4, one of the herbicidal active substances isolated from the culture broth of Streptomyces sp. KRA16-334, exhibited herbicidal activity against various weeds. The molecular formula of 334-W4 was determined to be C16H26N2O6, based on ESI-MS (m/z) and 1H and 13C NMR spectral data. It had molecular weight 365.1689 [M+Na] and 343.1869 [M+H], indicating the presence of the epoxy-β-aminoketone moiety based on HMBC correlations. Additionally, selective culture was possible depending on the addition of trifluoroacetic acid (TFA) during culture with GSS medium. Experiments confirmed that exposure of the KRA16-334 strain to UV irradiation (254 nm, height 17 cm) for 45 seconds improved the yield of the active substance (334-W4) by over 200%. As a result of examining yields of active materials of four mutants selected through optimization of culture conditions such as temperature, agitation, and initial pH, the yield of one mutant 0723–8 was 264.7 ± 12.82 mg/L, which was 2.8-fold higher than that of wild-type KRA16-334 at 92.8 ± 5.48 mg/L.


Introduction
Among various methods of weed control aiming at improving crop productivity, synthetic herbicides are widely used worldwide primarily due to their relatively low production costs and ability to provide effective control.However, various problems are emerging due to continuous use of organic synthetic herbicides.There problems include carcinogenic classification of glyphosate, a non-selective herbicide, safety issues such as human toxicity problems of paraquat, and emergence of resistant weeds due to long-term and continuous use of the same herbicide.Furthermore, due to commercialization of GMO (genetically modified organism) crops with glyphosate resistance, the use of herbicides other than glyphosate is decreasing.Even newly developed crop protection products are facing stricter global registration regulations in recent years.Therefore, the development of new herbicides with environmental friendliness and creative structures with new mode of action is needed to solve problems related to synthetic herbicides.However, since the development of 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors [1][2][3] such as benzobicyclon, mesotrione, and tefuryltrione, no herbicides with a new mode of action have been developed yet.Thus, it is necessary to move away from the existing imitation technology and develop a new concept herbicide.Unfortunately, no clear development results have been reported so far.In this situation, various attempts are being made worldwide to develop new herbicides, with a growing interest in natural products.Natural product-based herbicides are currently being actively explored through direct use of various natural substances or investigation of new substances with optimal chemical structures to act as lead compounds for developing novel synthetic compounds with unique functional mechanisms [4].
Actinomycetes produce a wide variety of physiologically active substances.Some of them are known to exhibit strong bioherbicidal activities.Herbicidal active substances discovered in actinomycetes so far include bialaphos produced by Streptomyces viridochromogenes or S. hygroscopicus [5,6] and hydantocidin produced by S. hygrioscopocus [7].In addition, there are thaxtomin A produced by S. scabies [8], actinonin produced by Actinomyces MG848-hf6, and albucidin, an active substance with herbicidal activity that causes bleaching from S. albus [9].Secondary metabolites produced by actinomycetes mainly consist of substances such as amino acids, peptides, nucleosides, macrolides, lactones, amides, and amines [10].Among these, compounds with potent herbicidal activities, such as Bialaphos [11,12] and Herbicidins [13,14], have been developed as commercial herbicides or are currently being studied.In addition, diverse structures and potent herbicidal activities of actinomycete secondary metabolites may offer not only direct applications as bioherbicides, but also potential for the development of synthetic herbicides with novel target sites.In particular, Streptomycetes sp. have the potential to produce new physiologically active substances.By exploring candidate materials as herbicides, new substances with different mechanisms of action compared to existing herbicides might be developed [15].Although active metabolites derived from actinomycetes show strong herbicidal activities, their productivity is low, which limits their industrial use.The productivity of actinomycete secondary metabolites is closely related to the optimization of culture conditions, including strain improvement.The technology for high-yield strain development and fermentation processes is recognized as the most important factor in the development of bioherbicides.
Therefore, this study was conducted to establish a foundation for the development of environmentally friendly bioherbicides.This was accomplished by isolating actinomycetes that showed strong herbicidal activity, identifying active compounds, and researching methods to improve the productivity of these compounds through culture optimization.

Isolation and culture of soil actinomycetes
Streptomyces sp.KRA16-334 isolate was separated and cultured using the method described previously [16].M3 medium (1% soytone, 1% glucose, 2% soluble starch, 0.3% CaCO 3 , 0.02% FeSO 4 , 0.1% antifoaming agent, 1 L water) was used for mass culture to isolate active substances and for selecting culture mutants.The culture was incubated on a rotary shaker (160 rpm) for 7 days at 27˚C.It was then centrifuged at 8,000 rpm for 10 minutes at 4˚C.After cells were removed, the culture filtrate was used to evaluate herbicidal activity.

In vivo assay for herbicidal activity
For the evaluation of herbicidal activity, seeds of five grass weeds (Sorghum bicolor, Echinochlia crus-galli, Agropyron smithii, Digitaria ciliaris, Panicum dichotomiflorum) and five broad-leaf weeds (Solanum nigrum, Aeschynomene indica, Abutilon avicennae, Xanthium strumarium, Calystegia japonica) were grown under greenhouse conditions using the methods described previously [17].The culture filtrate and active compounds of each concentration (14 mL per pot) were applied to the seedlings using a laboratory spray gun 12 days after sowing seeds.The herbicidal activity through foliar application was evaluated at 7 days after treatment by visually determining the percentage of injury (0%, no damage; 100%, complete control).All experiments were independently repeated three times.The mean percentage of injury was calculated based on three replicates.Distilled water was used as an untreated control.
The chemical structure of the active material was identified via molecular weight measurement using ESI-MS (SYNAPT G2, Waters) and NMR analysis of 1 H, 13 C NMR, 1H-1H COSY, HMBC, HSQC, NOESY, and ROESY (Bruker AVANCE HD 700 NMR spectrometer, 700 MHz for 1 H and 176 MHz for 13 C).NMR spectra were measured by dissolving in DMSOd 6 , CD 3 OD.Chemical shifts were referenced to the residual solvent signal (δ C 39.5 and δ H 2.50, δ C 49.15 and δ H 4.78).

Changes in yields of active compounds based on culture method
KRA16-334 was inoculated into M3 and GSS media (glucose 20, soluble starch 10, soybean meal 25, beef ext. 1, yeast ext.4, NaCl 2, KH 2 PO 4 0.25, CaCO 3 2 g/L) in 100 ml/500 ml baffled flasks and cultured in a rotary shaker (160 rpm) at 27˚C.After 3 days of incubation, 200 μL of trifluoroacetic acid (TFA) was added to each culture at 1-day intervals.The culture was then incubated for an additional 10 days.The yield of the active substance was determined and verified by conducting HPLC (Atlantis T3 column, 5 μm, 10x250 mm; WATERS) analysis of the culture filtrate using 0*100% acetonitrile containing 0.02% TFA over a period of 45 minutes at a flow rate of 0.8 mL/minute.Additionally, the herbicidal activity against D. ciliaris was evaluated.

UV mutagenesis for active compound 334-W4 yield-up
Mutagenesis for yield-up of an active strain was carried out according to the method described previously [18].KRA16-334 spore suspensions (1.2 x 10 3 ) were smeared onot Bennett agar plates, followed by exposure to UV light (254 nm, 17 cm distance) under dark conditions for 30 seconds to 45 seconds.Bacterial cells mutagenized by UV exposure were incubated at 27˚C for 5 days until colonies became visible.Survival and lethality rates were then assessed.Survival rate was calculated by dividing the number of colonies of UV-exposed plates by the number of colonies of plates without UV treatment.A UV exposure time that resulted in a 1% survival rate was determined through repeated experiments and subsequently utilized for mutagenesis to select high-yield mutants (S1 Table in S1 File).
Surviving mutant colonies were cultured in M3 liquid medium for 7 days following the previously described method (27˚C, 160rpm).Subsequently, cultures were centrifuged (8,000rpm, 10min) and the supernatant was utilized for activity and yield evaluation.The herbicidal activity of each mutant was evaluated using D. sanguinalis as previously described.Quantitative analysis was conducted to investigate the content of active compound 334-W4 in the culture under the following HPLC conditions: column: WATERS Atlantis T3 column (i.d.4.6 x 250 mm); mobile phase: aq.30% aqueous MeOH; flow rate: 0.8 mL/min; detection wavelength, 210 nm; and retention time: 30 minutes.After confirming the reproducibility of high-yield mutants first selected through subculture, mutants showing stable production were finally selected.

Culture optimization of selected mutants
Selected mutants used M3 liquid medium as basal medium.To establish optimal culture conditions, different culture temperatures, agitation speeds, and initial medium pH values were used.Content of active compound in culture broth was investigated by HPLC quantitative analysis.Mutants were incubated under the following conditions: 1) incubation temperature at 15, 20, 27, 30 or 35˚C; 2) agitation speed of 50 to 250 rpm; and 3) initial medium pH of 3, 5.5, 7, 8.5, or 10 adjusted using 1 N HCl and 1 N NaOH.Mutants were then cultured for 7 days.Culturing under each condition was replicated three times.

Statistical analyses
All treatments were statistically analyzed using one-way ANOVA (Analysis of Variance) in OriginPro 8.1 (OriginLab, 2021).Fisher's LSD test (p < 0.05) was used to compare means.

Classification and herbicidal activity of strain KRA16-334
The 16S rRNA sequence of strain KRA16-334 shared 99% similarities with 16S rRNA sequence of Streptomyces drozdowiczii strain NRRL B-24297 (Fig 1).Thus, it was named Streptomyces sp.KRA16-334.After culturing the KRA16-334 strain in M3 medium and evaluating herbicidal activities against 10 weeds using the culture filtrate, it was observed that the 2-fold dilution applied to leaves resulted in complete control of all weed species except for A. avicenae.In addition, when the culture filtrate was diluted four times and applied, it resulted in complete control (100%) for seven types of weeds except for S. bicolor, A. avicenae, and C. japonica.It also demonstrated more than 80% herbicidal activities against three weeds (Fig 2).Phytotoxic symptoms caused by KRA16-334 appeared within 24 h after culture broth foliage treatment, indicating that KRA16-334 acted very quickly.No weeds regenerated even at 7 days after treatment, suggesting a residual activity of KRA16-334.Major phytotoxic symptoms were wilting or leaf burn-down and stunting with eventual plant death.

Chemical properties and structure determination
Herbicide compounds 334-100M and 334-W4 were separated from the culture filtrate of Streptomyces sp.KRA16-334.334-100M was isolated from the 100% MeOH fraction when adsorbed to HP20 resin.It was purified by C 18 sep-pak cartridge and preparative high-performance liquid chromatography (prep HPLC).As a result of NMR analysis, 334-100M was identified as herboxidien, which contained a tetrahydropyran acetic acid moiety and a side chain with a conjugated diene.It had a molecular weight of 438.6 and a molecular formula of C 25 H 42 O 6 .Wideman et al. (1992) have reported that heboxidiene is a novel polyketide that exhibits potent herbicidal properties against several annual weed species [19].In addition, a study on the enhancement of herboxidiene productivity through gene regulation of Streptomyces chromofuscus ATCC 49982 has been reported [20].
334-W4 was isolated from the 50% MeOH fraction of HP20 CC.It is a novel herbicidal compound.It exhibited strong activities against broad-leaved weeds.Regarding chemical properties of 334-W4, it is a colorless oil that is soluble in H  quaternary carbons, and three carbonyl carbons (Table 1). 1  As a result of determining the structure of 334-W4 by 1D and 2D NMR and MS spectral analysis, it was identified as TMC-86A reported previously [17].TMC-86A isolated from fermentation broth of Streptomyces sp.TC 1084 has been reported as a novel 20S proteasome inhibitor with an epoxy-β-aminoketone moiety [21].However, its biological activity such as herbicidal activity has not been reported yet.

Changes in yields of active compounds based on culture method
To decrease the production of compound 334-100M reported to have herbicidal activity and to selectively cultivate the new active substance 334-W, KRA16-334 was cultured in the selective medium GSS.Comparison of culture broth using M3 and GSS media revealed that M3 medium resulted in significantly higher productivity of 334-100M than GSS medium.With the GSS medium, the yield of 334-W4 was increased, while that of 334-100M showed a relative decrease (Fig 4A and 4C).Additionally, it was confirmed that the productivity of the active material 334-100M was significantly reduced during culture when TFA was added to the medium throughout the culturing process.Particularly, adding TFA to the GSS medium on the 5th day resulted in no production of 334-100M, whereas the yield of 334-W4 was increased significantly (Fig 4).

Effects of culture conditions of selected mutants on yield of 334-W4 (TMC-86A)
Strain improvement strategies, especially mutagenesis and screening of hyper-producing mutants, are very important in the production of secondary metabolites during the fermentation process [22].In this study, Streptomyces sp.KRA16-334 was exposed to UV for different time periods.To optimize the quantity of mutagens, survival rate was calculated.When UV treatment was performed for 45 sec, its survival rate was 1.2 ± 0.57%.Screening of various mutants was performed based on 334-W4 (TMC-84A) yield.The wild type produced 69.9 mg/ L of 334-W4, while 45 sec UV-treated mutants produced up to 214.7 mg/L of 334-W4 (Fig 5).Among UV mutants tested, four mutants with high yields of 334-W4 were selected.The productivity of 334-W4 by culture temperature, agitation speed, and initial pH was then investigated.As a result of examining changes in yield according to culture temperature, the productivity of 334-W4 was excellent at 20˚C to 27˚C.It tended to decrease rapidly at 30˚C (Fig 6).In addition, the yield of the selected mutant was higher than that of the wild type at a stirring speed of 150 rpm or more and an initial pH of 7 to 8.5 (Fig 6).Streptomyces sp.KRA16-334 and its mutants were able to grow even under alkaline conditions of pH 10.As the agitation speed increased, the productivity of the active compound 334-W4 tended to increase.Among the four selected mutants, the yield of mutant 0723-8 (264.7 ± 12.82 mg/L) was 2.8 times higher than that of the wild type KRA16-334 (92.8 ± 5.48 mg/L).It was the highest at a temperature of 27˚C, an agitation speed of 250 rpm, and an initial pH of 8.5.

Discussion
Secondary metabolites produced by Streptomyces sp. are being studied as potential bioherbicides or herbicide adjuvants in order to develop environmentally friendly agents that can be easily degraded with a low toxicity.In addition, isolation and structural identification of native herbicide-active compounds from Streptomyces sp. have been proven to be an effective method for discovering new leads in herbicide development.Therefore, numerous isolation and screening attempts have been made to discover microbial metabolites with bioherbicidal potential for Streptomycetes.In this study, we selected KRA16-334, which exhibited herbicidal activities against several weeds, and identified it using 16S rRNA gene sequencing analysis.Among molecular characterization techniques, 16S rRNA gene sequence analysis is a more suitable and time-saving approach to identify Streptomyces [23].The 16S rRNA gene sequence of the selected KRA16-334 strain shared 99% sequence similarities with the16S rRNA gene of S. drozdowiczii strain NRRL B-24297.Thus, it was named Streptomyces sp.KRA16-334.Characteristics of herbicidal activity, include wilting, leaf burn-down, stunting, and ultimately plant death, were then observed after applying KRA16-334 culture filtrate to leaves.334-100M and 334-W4 were herbicidal active substances isolated from the fermentation broth of Streptomyces sp.KRA16-334.334-100M was heboxidiene with a molecular weight of 438.6 and a molecular formula of C25H42O6.Wideman et al. (1992) have reported that heboxidiene is a novel polyketide that exhibits potent herbicidal properties against several annual weed species [19].In addition, Jha et al. (2014) have reported that herboxidiene productivity can be enhanced through gene regulation of Streptomyces chromofuscus ATCC 49982 [20].334-W4 was identified as TMC-86A having a molecular formula of C16H26N2O6.TMC-86A, a novel 20S proteasome inhibitor with an epoxy-β-aminoketone moiety, has been reported previously [21].However, its biological activity such as herbicidal activity has not been reported yet.
Addition of TFA to culture medium during culturing had a significant impact on the production of active compounds (Fig 4).The productivity of 334-100M with the M3 medium was significantly higher than that with the GSS medium.With the GSS medium, the yield of 334-W4 was increased whereas that of 334-100M showed a relative decrease.Adding TFA to the GSS medium on the 5th day resulted in no production of 334-100M, although the yield of 334-W4 was increased significantly.These findings suggest that manipulating culture conditions, specifically by using GSS medium and adding TFA, can influence the production of active substances, offering insights for potential herbicidal applications.The ability of Streptomyces sp. to produce active substances can be greatly increased or completely lost under various nutritional and culturing conditions.Thus, it is not a fixed characteristic [24].
Productivity improvement of secondary metabolites produced by Streptomyces sp.depends on optimization of culture conditions, induction of high-yield mutations, and metabolic engineering.In particular, the selection of mutants and the screening of overproducing mutants through appropriate strain-improvement techniques are critical for the production of secondary metabolites during the fermentation process.To have enough production of the herbicidal compound 334-W4, the most important and convenient physical method for obtaining a wide spectrum of mutations was using UV radiation.To achieve powerful and effective mutation selection through UV radiation, a high lethality rate is required [25].Therefore, four mutant strains with increased productivity of 334-W4 were selected through 45 seconds of UV irradiation at a height of 17 cm.Regarding the influence of culture temperature, the mutant strain showed excellent productivity of 334-W4 at 20˚C and 27˚C.However, a significant decrease in productivity was observed at temperatures below 15˚C and above 30˚C.As a result, mutants exhibited a restricted range of culturing temperatures for the production of 334-W4.It was determined that the optimal culture temperature for achieving maximum production of 334-W4 was 27˚C.The optimal initial pH for the production of secondary metabolites by Streptomyces sp. was found to be neutral pH, which was reported in several studies [26,27].Mutants selected through experiments were found to produce the optimal active substance when the pH level was between 7 and 8.5.It was also observed that the maximum productivity of the active substance was achieved at an agitation speed of 250 rpm.This indicates that the production of the active substance 334-W4 by mutants is related to an increase in agitation speed.Agitation was found to have a direct impact on the mixing of nutrients and the amount of dissolved oxygen in the medium, which is crucial for the production of 334-W4.Through studies aimed at removing toxic substances by modifying cultural methods and increasing the yield of the active substance 334-W4 through UV mutation and cultural optimization, Streptomyces sp.KRA16-334 culture medium is believed to offer an opportunity for the development of bioherbicides for direct use.Additionally, herbicide compound 334-W4 is considered a promising candidate for the development of novel herbicides and synthetic herbicides with new mechanisms.

Conclusions
Structural diversity and potent herbicidal activity of actinomycete secondary metabolites not only can be directly used as herbicides, but also can provide opportunities for the development of synthetic herbicides targeting new sites.In this study, we isolated and cultured Streptomyces sp.KRA16-334, which exhibited strong herbicide activities.Yield improvement and selective culturing of the herbicidal compound 334-W4 were achieved through UV mutation, culture optimization, and modification of culture medium.The yield of the active compound 334-W4 from Mutant 0723-8 (264.7 ± 12.82 mg/L) selected through UV mutation increased by 2.8-fold compared to that of the wild type KRA16-334 (92.8 ± 5.48 mg/L).The highest yield was observed at a temperature of 27˚C, an agitation speed of 250 rpm, and an initial pH of 8.5.In addition, the strain 334-W4 was able to be selectively cultured by adding TFA during culturing in GSS medium.Therefore, the culture broth of Streptomyces sp.KRA16-334 can be utilized as a safe bioherbicide.In addition, the herbicidal compound 334-W4 can provide an opportunity for the development of new herbicides.